The technical field of the present invention is that of probes constituted by labelled, mono-strand DNA or RNA nucleic acid sequences.
Such probes are well known in the state of the art and may be obtained by various routes particularly by genetic engineering or by manual or automatic direct synthesis.
These nucleic acid sequences have the property of being matched and of forming hybrids with complementary DNA sequences, as the case may be, denaturated previously, if the latter were initially bicatenary or mRNA. This denaturation can be done after incubation in a medium of high ionic strength and at high temperature or in a basic medium. These hybrids are then detectable.
The detection of the hybrids can be done by different methods. The probe may be labelled by one of the known methods for the labelling of nucleic acid probes. It may be radioactive labelling, for example with phosphorus 32, or in the case of a cold probe, non-radioactive labelling, for example enzymatic, as is also known. In certain cases, the probe is not labelled during its use proper, but modified chemically to be detectable after hybridization, for example with biotin.
More precisely, the present invention relates to nucleic acid probes enabling the detection of different types of human papilloma virus by hybridization with the DNA of the virus. It relates particularly to eight types of human papilloma virus (HPB) of which all the DNA sequences are known today, namely the types HPV1a, HPV5, HPV6b, HPV8, HPV11, HPV16, HPV18 and HPV33.
10% of women are infected with the HPV virus; the virus is located in the cervical cells and leads in a certain number of cases to dysplasic and/or malignant lesions of the cervix, Among the various types of HPV virus, HPV16 and HPV18 seem most frequently associated with these lesions.
HPVs are characterized by a circular double strand DNA genome, about 8000 pairs of bases long, wrapped in a protein capsid 60 nm in diameter. The virus subsists in non-malignant lesions in the unintegrated episome state. In these cases disturbance of the cellular differentiation is observed and the production of viral particles at late stages of the differentiation. When uterine carcinoma is established, a variable integration of certain sequences of viral DNA in the cellular DNA is observed.
The detection of HPV virus in the cervix is carried out by the use of molecular probes constituted by isotopically cloned and labelled viral DNA fragments.
Within the framework of epidemiological studies and routine diagnosis of infections by HPV, it is useful to develop a rapid simple specific and sensitive method based on the use of synthetic DNA probes labelled non-isotopically and usable in situ. To this end, there are identified, at the level of the DNA, specific regions of different types of HPV virus to deduce therefrom complementary homologous synthetic probes. The nucleotide sequences of HPV1, HPV5, HPV6, HPV11, HPV16, HPV18 and HPV33 are known.
It is an object of the present invention to provide probes characteristic of all the various types of HPV virus particularly mentioned above which are effective, that is to say probes having the longest possible nucleic acid sequences and which are common to the various types of HPV virus.
Another object of the present invention is also to provide specific probes for each of the various types of HPV virus and which ensure stable matching.